Characterization of Escherichia coli dinJ-yafQ Toxin-Antitoxin System Using Insights from Mutagenesis Data

Abstract

Escherichia coli dinJ-yafQ operon codes for a functional toxin-antitoxin (TA) system. YafQ toxin is an RNase which, upon overproduction, specifically inhibits the translation process by cleaving cellular mRNA at specific sequences. DinJ is an antitoxin and counteracts YafQ-mediated toxicity by forming a strong protein complex. In the present study we used site-directed mutagenesis of YafQ to determine the amino acids important for its catalytic activity. His50Ala, His63Ala, Asp67Ala, Trp68Ala, Trp68Phe, Arg83Ala, His87Ala, and Phe91Ala substitutions of the predicted active-site residues of YafQ abolished mRNA cleavage in vivo, whereas Asp61Ala and Phe91Tyr mutations inhibited YafQ RNase activity only moderately. We show that YafQ, upon overexpression, cleaved mRNAs preferably 5= to A between the second and third nucleotides in the codon in vivo. YafQ also showed RNase activity against mRNA, tRNA, and 5S rRNA molecules in vitro, albeit with no strong specificity. The endoribonuclease activity of YafQ was inhibited in the complex with DinJ antitoxin in vitro. DinJ-YafQ protein complex and DinJ antitoxin alone selectively bind to one of the two palindromic sequences present in the intergenic region upstream of the dinJ-yafQ operon, suggesting the autoregulation mode of this TA system.

Identification and Characterization of Type II Toxin-Antitoxin Systems in the Opportunistic Pathogen Acinetobacter baumannii

“…The E. coli BW25113 F= pUHEcat-"antitoxin" pBAD-"toxin" strains were grown in LB medium at 37°C until an OD 600 of 0.1 was reached, and arabinose was then added to a final concentration of 0.2% for toxin induction. The samples were collected before and after different times of induction and subjected to RNA extraction with hot phenol, as described previously (26). Fifteen micrograms of RNA per sample was loaded onto a 2% agarose gel.…”

Relaxed Cleavage Specificity within the RelE Toxin Family

“…While R61 is conserved in the 8 sequences, Y87 and R81 are less conserved (5 and 3, respectively, out of 8). The M. avium RelE, RelE Mav , possesses a phenylalanine at the position corresponding to Y87, similar to the H. pylori HP0894 RelE-like toxin (F88) and E. coli YafQ (F91) (28,37). In addition, RelE Mav contains part of an HP0894 motif involved in substrate recognition (E107, L108, and F109) (28).…”

Activation of Toxin-Antitoxin System Toxins Suppresses Lethality Caused by the Loss of σ E in Escherichia coli

“…Also, some of these toxins might need to cooperate to suppress the E essentiality. Unlike HicA, HigB and YafQ are ribosome-dependent mRNA interferases (45,46,53,54), indicating that an mRNA cleavage mechanism does not define the ability of toxins to suppress the E essentiality. Previous primer extension analyses of HicA and YafQ did not reveal common target motifs (14,45,46); HicA apparently has no obvious consensus recognition motif, whereas YafQ preferentially cleaves mRNA at the 5= side of adenine residues (46).…”

“…Unlike HicA, HigB and YafQ are ribosome-dependent mRNA interferases (45,46,53,54), indicating that an mRNA cleavage mechanism does not define the ability of toxins to suppress the E essentiality. Previous primer extension analyses of HicA and YafQ did not reveal common target motifs (14,45,46); HicA apparently has no obvious consensus recognition motif, whereas YafQ preferentially cleaves mRNA at the 5= side of adenine residues (46). However, it is possible that more accurate identification of the cleavage specificities of these toxins by using alternative techniques, such as transcriptome sequencing (RNA-seq) (28), to identify their common targets could help us to understand the mechanisms by which these TA systems function in response to envelope stress.…”